Multiphase systems are rapidly becoming popular in extraction processes as well as in novel reaction processes.
Such systems are particularly attractive, for example, in extraction of protein. While a number of efficient methods for protein purification with high specificity are available for downstream processing, few methods can be employed for large volume, upstream applications. As a result, much of the protein is lost in early stages of processing. Techniques such as ultrafiltration and various methods of precipitation have been used, but offer only minimal selectivity based on size range, isoelectric point or other properties which are common to a large number of proteins in a mixture. Two-phase extraction systems, on the other hand, offer both high volume production as well as specificity.
Aqueous two-phase systems have been described for the large-scale isolation and purification of proteins and biological analysis. Generally, phase separation is a phenomenon that occurs when two solutions of water-soluble polymers are mixed. Instead of using two polymers, a polymer and a salt solution can be used to form an aqueous two-phase solution. The most commonly used polymers are polyethylene glycol (PEG) and dextran. However, many other polymers have been shown to form two-phase systems. Among the polymer-salt systems, PEG/potassium phosphate and PEG/magnesium sulfate are most frequently used. E. Andersson and B. Hahn-Hagerdal, "Bioconversions in Aqueous Two-phase Systems," Enzyme Mircrob Tchnol., 12: 242-254 (1990).
The technique of two-phase extraction in protein separation involves contacting the two liquid phases with a mixture of proteins. The properties of the phases are such that the desired proteins partition preferably into one of the two layers. These properties may include hydrophobicity, pK.sub.a, size, density or any of a number of affinities for the components of a particular phase. Consequent separation of the phases results in containment of the protein(s) of interest in one of the phases. The efficiency of the separation is dependent on the partition coefficients of the proteins, ideally with the protein of interest having the highest partition coefficient of the mixture.
The development of two-phase systems has included the use of affinity ligands to significantly enhance selectivity of protein partitioning. Such affinity ligands include any of a number of entities that preferentially bind a component of interest.
Reactive bioconversions in aqueous two-phase systems have also been described. Biocatalysts (microorganisms, enzymes) can be sensitive to pH, temperature, ionic strength and organic solvents. They are also often expensive. In a chemical/biochemical process, it is therefore desirable to protect them and to be able to reuse them. It is also desirable to obtain a product stream which is as pure and as concentrated as possible.
Extractive bioconversion utilizing two liquid phases with the biocatalyst present in only one of the phases, makes it possible to recover the product from the biocatalyst-free phase. Starch hydrolysis, for example, is a major enzyme-catalyzed process in industrial operation today. The feasibility of carrying out enzymatic starch hydrolysis in aqueous two-phase systems, performing the process in one of the phases and continuously extracting the product to the other phase, has been reported. N. Larson et al. , "Integration of Bioconversion and Downstream Processing: Starch Hydrolysis in an Aqueous Two-Phase System," Biotechnology and Bioengineering 33:758-786 (1989).
Recently the principles and applications of two-phase systems have been used in conjunction with reversed micellar technology. M. E. Leser and P. L. Luisi, Chimia 44:270-282 (1990); M. Dekker, Anal. Brochem. 178:217-226 (1990); T. A. Hatton, et al. eds., Surfactant-Based Separation Processes, Marcel Dekker, Inc., 55-90 (1989). Reversed micelles may be described as spontaneously formed spherical aggregates of surfactant molecules in organic solvents. These colloidal systems are formed upon addition of a small volume of water to a much larger volume of immiscible organic solvent(s) containing a surfactant agent.
Reversed micelles, or water-in-oil (W/O) microemulsions, can thus be seen as water droplets solubilized in apolar solvents by virtue of a layer of surfactant molecules. The polar heads of the surfactant molecules are directed toward the interior of the micelles, creating a polar core, where water is localized (i.e., a "water pool").
The water pools of reverse micelles are capable of solubilizing many large hydrophilic molecules including proteins. Protein extraction in a reversed micellar system has been reported, for example, by G. A. Ayala et al. in "Protein Extraction and Activity in Reverse Micelles of a Nonionic Detergent," Biotechnology and Bioengineering, 39:806-814 (1992).
Another important feature of reversed micelles is the differential nature of the polar core and the surrounding medium, especially when composed of hydrocarbon. In designing organic solvent/water systems for biocatalytic reactions, one has to consider the toxicity of the organic solvent for the biocatalyst. In this regard, it has been shown in the last few years that enzymes can be hosted in the water pool of reverse micelles without loss of activity. This observation has attracted considerable interest in the biotechnological areas because of the potential of carrying out biocatalysis in essentially organic solvents as well as protein separation.
The incorporation of enzymes in reversed micelles has thus become a popular method of exploiting their catalytic properties in a predominantly organic environment. The water soluble enzymes are retained in the water droplets, where they maintain their catalytic properties as in bulk aqueous phase. The interest in using micelles as hosts for enzymes lies primarily in the advantages of the continuous organic phase. Many industrially important reactions are limited to organic media because of solubility. Solutes in the organic phase of the micelle system partition into the water pools where they are converted to product by the enzyme. The products, in turn, partition back into the organic bulk phase. In this way, high concentrations of substrate and product can be admitted without inhibitory effects to the enzyme.
Reversed micelles to date have found a number of applications, including some commercial uses. Jorba et al., in "Optimization and Kinetic Studies of Enzymatic Synthesis of Ac-PHE-Leu-NH Subgroup Two in Reversed Micelles," Enzyme Microbiology Technology, 14 (117-124) (February 1992), for example, describe the application of reversed micellar systems in peptide synthesis.
L. Qwang and T. F. Yen, in "Sulphur Removal From Coal Through Multiphase Media Containing Biocatalysts," Journal Chem. Tech. Biotechnol., 48:71-79 (1990), disclose that the sulphur content in coals can be reduced through multiphase media containing biocatalysts such as bacterial cells and cell-free enzyme extracts in a batch process. The results showed that the multiphase processes using biocatalysts have an efficient sulphur removability and a shorter reaction time than conventional microbial processes. Tween 80 was used as a surfactant in the study.
N. Phammatter et al., in "Solubilization and Growth of Candida Pseutotropicalis in Water-In-Oil Microemulsions," Biotechnology and Bioengineering, 40:167-172 (1992), described the growth rate of candida pseudotropicalis in water-in-oil microemulsions in a hexadecane solution containing Tween 85 and Span 80 as surfactant with a limited amount of water therein.
While multiphase extractive and reactive techniques are being demonstrated in principle, demand increases for process systems for larger scale use. Therefore, an important step in developing a process which utilizes a multiphase technique, and particularly reversed micellar systems, for a commercial operation is the design of an efficient continuous system. Several attempts have been made to achieve this goal.
P. Luthi and T. A. Hatton, "Recovery of Biocatalysis Products from Reversed Micellar Reaction Media: A Preliminary Evaluation of Membrane Extractors," Bioseparation, 2:5-14 (1991), for example, demonstrated that ultrafiltration membranes of sufficiently low molecular weight cutoff can be used to retained reversed micelles and their hosted enzymes, while permitting the recovery of lipophilic products of enzymatically-catalyzed, synthesis reactions in a stripping solution on the other side of the membrane. Calculations indicated that hollow fiber membranes having the same rejection characteristics and solvent resistance as the flat sheet membranes, may provide an attractive an efficient means for the recovery of these biosynthesis products.
Chiang and Tsai, in "Application of a Recycled Dialysis System in a Reversed Micellar Reactor," J. Chem. Tech. Biotechnol., 54: 27-32 (1992), disclose the use of a dialysis membrane reactor for integrating reaction and product recovery in reversed micellar systems.
These hollow fiber reactors, recycle dialysis reactors and stirred tank reactors, however, do not provide a satisfactory continuous operation for multiphase systems. The cost and design complexity of the first two reactors render their use in an industrial continuous process unattractive. While the stirred tank reactor design may provide simplicity of design, the requirement of additional equipment to effect separation results in undesirable expense. It is thus desirable to develop multiphase systems that requires only a single unit to effect (1) extraction and/or reaction as well as (2) separation.
Continuous centrifugation has been used in the past for effecting continuous liquid-liquid extraction in a single operational unit.
Also, Van Wie et al., in "A Novel Continuous Centrifugal Bioreactor for High-Density Cultivation of Mammalian and Microbial Cells," Biotechnology and Bioengineering, 38:1190-1202 (1991) and U.S. Pat. No. 4,939,087 disclose a continuous centrifugal bioreactor to study the growth and productivity of dense suspension cultures. The reactor was used in both fermentation and mammalian cell cultivation processes. Van Wie et al. disclose that their reactor can maintain high cell concentrations in a well mixed environment without appreciable cell elutriation. Cells are maintained in the reactor at a desired density by balancing the centrifugal forces with the drag across cell surfaces as media flow is directed radially inward. The reactor is of a tapered shape for better cell retention by providing a large variation in superficial velocity, being of high magnitude at the cone entrance and relatively small values closer to the center of rotation. Because of the angle of taper, the reduction in velocity compensates for the reduced centrifugal forces at small radial distances from the center of rotation. In effect, the centrifugal bioreactor of Van Wie et al. acts to immobilize the cells.
Until the present, however, centrifugation has not been used to effect a multiphase catalytic reaction. Nor has centrifugation been used to effect multiphase extraction of proteins. Nor has centrifugation been used in connection with high affinity extraction means.
It is thus an object of the present invention to provide a centrifugal system for single-stage, multiphase reaction/separation processes. It is also an object of the present invention to provide a continuous centrifugal system for single-stage, multiphase extraction/separation utilizing extraction and high-affinity extraction means in one phase.